I plan to utilize genetic and biochemical techniques to examine the role of DNA polymerase I in DNA polymerization. All eucaryotic and procaryotic DNA polymerizing enzymes form phosphodiester bonds via the addition of 5'-triphosphates to 3'-hydroxyl groups of growing chains. DNA polymerase I carries out this reaction and in addition catalyzes 5' to 3' to 5' nucleolytic attack on pre-existing DNAs and DNA-RNA hybrid structures. Despite substantial documentation of the molecule's enzymatic characteristics, knowledge of the primary and secondary structure of the protein is incomplete. Our ignorance of the amino acid sequence and the conformation of the enzyme's active sites reflects the fact that the molecule is an unusually large polypeptide and is available from natural sources in limited quantities. As a result structural studies of intact native enzyme are complex and expensive. The isolation of a series of mutational alleles of the structural gene for DNA polymerase I has made it possible to construct a genetic fine-structure map of the polA gene. Correlation of enzymatic defects with genetic map position has been made and allows inference of the direction of transcription of the cistron. I plan to extend these studies to construct a more complete map and to utilize individual mutant enzymes to allow ordering of individual cyanogen bromide or tryptic peptides in the DNA polymerase finger print. Part of the plan of these experiments involves construction of specialized transducing phage to help amplify the polA gene products.